Astrocytes amplify neurovascular coupling to sustained activation of neocortex in awake mice

Functional hyperemia occurs when enhanced neuronal activity signals to increase local cerebral blood flow (CBF) to satisfy regional energy demand. Ca2+ elevation in astrocytes can drive arteriole dilation to increase CBF, yet affirmative evidence for the necessity of astrocytes in functional hyperemia in vivo is lacking. In awake mice, we discovered that functional hyperemia is bimodal with a distinct early and late component whereby arteriole dilation progresses as sensory stimulation is sustained. Clamping astrocyte Ca2+ signaling in vivo by expressing a plasma membrane Ca2+ ATPase (CalEx) reduces sustained but not brief sensory-evoked arteriole dilation. Elevating astrocyte free Ca2+ using chemogenetics selectively augments sustained hyperemia. Antagonizing NMDA-receptors or epoxyeicosatrienoic acid production reduces only the late component of functional hyperemia, leaving brief increases in CBF to sensory stimulation intact. We propose that a fundamental role of astrocyte Ca2+ is to amplify functional hyperemia when neuronal activation is prolonged.

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Replication Source data are provided with this paper. Raw image files are stored on servers at Hotchkiss Brain Institute owing to their large size. These raw images can be provided from the corresponding author upon request.
n/a n/a n/a n/a Based on previous effect sizes and error, n of 5-10 is typically sufficient power to test for significance in brain slices (lnstitoris, A. et al., J. Cereb. Blood Flow Metab. 35, 1411-1415(2015; Rosenegger et al., J. Neurosci. 35, 13463-13474 (2015). For in vivo observational experiments ( Fig. 1-3, Supplementary Fig. 1-3 and 7-8) we expected to be able to collect data from 2 vessels per mouse on average, based on the time period an awake mouse tolerates the experiment.
No data were excluded.
In Figure 1E, astrocyte endfoot and process Ca2+ and arteriole dilation response latencies match the findings described in a previous paper by the Gordon lab, using an identical but different 2-photon fluorescent microscopy rig. See Tran, C.H. et al., Neuron 100, 1133-1148.e3 (2018.
In the previous submission of the manuscript the CalEx experiments were carried out using an acute cranial window preparation and a less optimal viral control, astrocyte targeted tdTomato. The main effect of CalEx reducing the late phase of arteriole dilation to 30sec whisker stimulation was the same. However, in astrocytes expressing tdTomato, the measured astrocyte Ca2+ signals were reduced, most likely as a result of tdTomato quenching the fluorescent light emitted by GCaMP. This rendered the effect of CalEx on attenuating astrocyte Ca2+ to be less significant than in the published CalEx experiment.
The novel kinetics of penetrating arteriole responses has been reproduced by our lab using intrinsic optical imaging, which is available as a pre-print on BioRXiv (doi: https://doi.org/10.1101/2021.09.15.460557).
Vascular and cellular Ca2+ responses of short electrical stimulation of rat brain slices have been previously identified and reliably reproduced in lnstitoris, A. et al., J. Cereb. Blood Flow Metab. 35, 1411-1415(2015.